All-trans-retinoic acid (ATRA)-grafted polymeric gene carriers for nuclear translocation and cell growth control

Polyethyleneimine (PEI)-g-All-trans-retinoic acid (ATRA) (designated as PRA) was synthesized as a gene carrier. ATRA at its low concentration is known to be linked to nuclear translocation and cell cycle control (either proliferation or growth arrest) depending on its binding protein in cells. The cytotoxicity of PRA conjugates was lower than that of PEI and was gradually reduced as increasing ATRA graft ratios. The resulting nanosized and positively charged PRA/pDNA complexes showed lower transfection efficiency than the PEI/pDNA complexes (N/P = 10) against NIH3T3 which is less sensitive to ATRA in cell growth and more sensitive HeLa cells. However, when a mixed gene complex of PEI and PRA was applied in an effort to reduce the ATRA contents, their NIH3T3 transfection evidenced effective nuclear translocation and induced 2- to 4-fold better transfection efficiency as compared with the PEI/pDNA complexes. When the PEI/pDNA complexes were utilized to transfect HeLa cells, free ATRA treatment reduced their cellular uptake and transfection efficiency. These findings show that the NIH3T3 cells against ATRA-mediated growth arrest would not damage the PRA-mediated transfection enhancement resulting from the facilitated nuclear translocation of polyplexes or pDNA. The more ATRA-sensitivity in growth arrest of HeLa cells would reduce the transfection efficiency of ATRA-incorporated polyplexes. The transfection capability of gene by newly synthesized PRA conjugates to cells is differentiated by their ATRA-sensitivity to nuclear translocation and cell growth control.     

Self-assembled glycol chitosan nanoparticles for the sustained and prolonged delivery of antiangiogenic small peptide drugs in cancer therapy

Antiangiogenic peptide drugs have received much attention in the fields of tumor therapy and tumor imaging because they show promise in the targeting of integrins such as alpha(v)beta(3) on angiogenic endothelial cells. However, systemic antiangiogenic peptide drugs have short half-lives in vivo, resulting in fast serum clearance via the kidney, and thus the therapeutic effects of such drugs remain modest. In this study, we prepared self-assembled glycol chitosan nanoparticles and explored whether this construct might function as a prolonged and sustained drug delivery system for RGD peptide, used as an antiangiogenic model drug in cancer therapy. Glycol chitosan hydrophobically modified with 5beta-cholanic acid (HGC) formed nanoparticles with a diameter of 230 nm, and RGD peptide was easily encapsulated into HGC nanoparticles (yielding RGD-HGC nanoparticles) with a high loading efficiency (>85%). In vitro work demonstrated that RGD-HGC showed prolonged and sustained release of RGD, lasting for 1 week. RGD-HGC also inhibited HUVEC adhesion to a beta ig-h3 protein-coated surface, indicating an antiangiogenic effect of the RGD peptide in the HGC nanoparticles. In an in vivo study, the antiangiogenic peptide drug formulation of RGD-HGC markedly inhibited bFGF-induced angiogenesis and decreased hemoglobin content in Matrigel plugs. Intratumoral administration of RGD-HGC significantly decreased tumor growth and microvessel density compared to native RGD peptide injected either intravenously or intratumorally, because the RGD-HGC formulation strongly enhanced the antiangiogenic and antitumoral efficacy of RGD peptide by affording prolonged and sustained RGD peptide delivery locally and regionally in solid tumors.     

An endosomolytic Tat peptide produced by incorporation of histidine and cysteine residues as a nonviral vector for DNA transfection

Peptides as functional biomaterials offer the possibility of incorporating various biological activities required for different biomedical applications. Here, we take advantage of this property of peptide materials and design a DNA delivery vector equipped with multiple functions critical to efficient gene transfection. The Tat peptide, a cationic cell-penetrating peptide, is known to enhance the cellular uptake of a large variety of molecules such as drugs and proteins. However, the application of the Tat peptide in DNA delivery is limited by the inability to release DNA in endosomes and the instability of peptide/DNA complexes. We incorporate in the Tat sequence histidine and cysteine residues that are able to promote endosomal escape of DNA and protect DNA in the extracellular environment. We observe up to 7000-fold improvement in gene transfection efficiency by a modified Tat peptide covalently fused with 10 histidine residues (Tat-10H) over the original Tat peptide. After incorporating two cysteine residues into the Tat-10H design, the resulting bis(cysteinyl) histidine-rich peptide is more effective than the Tat-10H peptide, because interpeptide disulfide bonds form by air oxidation upon binding to DNA, leading to enhanced stability of peptide/DNA complexes. These findings demonstrate the feasibility of using multi-functional peptide materials to extend the applications of the Tat vector to efficient gene delivery.     

A novel transfecting peptide comprising a tetrameric nuclear localization sequence

The transport of exogenous DNA into the nucleus of eukaryotic cells is a prerequisite for successful gene delivery. To favor nuclear transport we synthesized a tetramer of the nuclear localization signal (NLS) of the SV40 large T-antigen as a novel nonviral gene delivery vector. This 4.4-kDa lysine-rich peptide (NLSV404) binds and compacts DNA by electrostatic interaction and forms stable polyplexes. Apart from its sequence-specific potency to mediate nuclear accumulation of conjugated albumin, NLSV404 also displays properties of nuclear transport for plasmid DNA as confirmed by fluorescence in situ hybridization. Further, NLSV404 polyplexes are shown to efficiently transfect various cell lines such as 16HBE14o–, HeLa S6, and Cos7 cells. NLSV404 polyplexes displayed at least 20-fold higher transfection rates than analogous polyplexes formed by the nuclear transport-deficient mutant sequence cNLS. Using growth-arrested cells, NLSV404 complexes were at least 100-fold more efficient than cNLS complexes. Combination of NLSV404 peptide but not of cNLS peptide with preformed polyethylenimine and dendrimer DNA complexes resulted in a strong increase in transfection efficiency. Incubation of cells prior to transfection with NLSV404 polyplexes with excess free peptide NLSV404 but not with cNLS resulted in a dose-dependent dramatic decrease in the transfection rate, suggesting a sequence-specific competitive inhibition. These results indicate that NLSV404 mediates nuclear accumulation of transfected plasmid DNA and that it can be a highly useful component of nonviral gene vectors.Abbreviations BSA-BODIPY Fluorescence-labeled bovine serum albumin - EGFP Enhanced green fluorescent protein - FISH Fluorescent in situ hybridization - NLS Nuclear localization signal - PEI Polyethylenimine - SV40 Simian virus 40     

Self-assembled ternary complexes of plasmid DNA, low molecular weight polyethylenimine and targeting peptide for nonviral gene delivery into neurons

Chemical conjugation of targeting ligands to polycation/plasmid DNA complexes has been widely used to improve the transfection efficiency of nonviral gene delivery vectors. However, conjugation reactions may reduce or even inactivate the biological activities of chemically sensitive moieties, such as proteins and peptides. Here we describe a new method for introducing targeting ligands into nonviral vectors, in which ternary complexes are formed via charge interactions among polyethylenimine (PEI) of 600Da, plasmid DNA and targeting peptides with positively charged DNA-binding sequence. Owing to the nerve growth factor (NGF) loop 4 hairpin motif in the targeting peptide, these ternary complexes are capable of mediating gene delivery efficiently and specifically into cells expressing the NGF receptor TrkA. In in vitro experiments, the complexes improved luciferase reporter gene expression by up to 1000-fold while comparing with that produced by complexes with nontargeting control peptide. In an in vivo experiment, the ternary complexes with the targeting peptide was 59-fold more efficient than the control ternary complexes in transfecting dorsal root ganglia (DRG), the peripheral nervous sites with TrkA-expressing neurons. In a cell viability study, the ternary complexes were remarkably different from DNA complexes by PEI of 25 kDa, the gold standard for nonviral gene carriers, displaying no toxicity in tested neuronal cells. Thus, this study demonstrates an alternative method to construct nonviral delivery system for targeted gene transfer into neurons.     

Arg-Gly-Asp (RGD) peptide ameliorates carbon tetrachloride-induced liver fibrosis via inhibition of collagen production and acceleration of collagenase activity

Liver cirrhosis is caused by a relative imbalance between synthesis and degradation of collagens. Arg-Gly-Asp (RGD) peptide is a major adhesive domain of several extracellular matrix (ECM) components, such as that involved in the binding of fibronectin to the alpha5beta1 integrin receptor. We previously reported that RGD peptide increased the expression of matrix metalloproteinase in hepatic stellate cells (HSCs) which play a major role in hepatic fibrosis. We evaluated whether RGD-peptides inhibit the progression of liver fibrosis in an animal model of carbon tetrachloride-induced hepatotoxicity. RGD peptide (GRGDS) (1 mg/kg body weight) was injected intraperitoneally (i.p.) 3 times a week for one month. The group treated with control peptide (GRGES) showed pathologically typical hepatic fibrosis, while the RGD-treated group showed minimal fibrotic changes. The liver contents of collagen and hydroxyproline in the RGD-treated group was significantly lower than that of the control group. Collagenase activity measured in liver homogenates was significantly higher in the treated group than in the control group. In an in vitro study using TWNT-4 cells derived from human HSCs, RGD peptide (100 mug/ml) reduced the expression of type I collagen and tissue inhibitor of matrix metalloproteinase-1, and increased that of matrix metalloproteinase-1. These results indicated that RGD peptides inhibited liver fibrosis associated with both decreased collagen production and increased collagenase acitivity, and suggested that RGD peptide might be useful for the therapy of hepatic fibrosis.     

Development of CD8 alpha/beta + TCR alpha beta intestinal intraepithelial lymphocytes in athymic nu/nu mice and participation in regional immune responses.

On the basis of the CD8 coreceptor expression, T-cell receptor (TCR)alpha beta-bearing intestinal intraepithelial lymphocytes (i-IEL) segregate into two populations. The CD8 alpha alpha + TCR alpha beta i-IEL develop thymus independently, whereas the CD8 alpha beta + TCR alpha beta i-IEL are generally considered to be thymus dependent. Flow cytometry analysis revealed a distinct population of CD8 alpha beta + TCR alpha beta i-IEL in individual athymic nu/nu mice. The i-IEL encompassing CD8 alpha beta + TCR alpha beta cells expressed potent cytolytic and interferon-gamma-producing activities. These findings demonstrate that CD8 alpha beta + TCR alpha beta i-IEL can develop in nu/nu mice independently from a functional thymus and suggest that these cells, directly or indirectly, perform biological functions in the gut.     

Gene transfection of hyperbranched PEI grafted by hydrophobic amino acid segment PBLG

The complex copolymer of hyperbranched polyethylenimine (PEI) with hydrophobic poly(gamma-benzyl L-glutamate) segment (PBLG) at their chain ends was synthesized. This water-soluble copolymer PEI-PBLG (PP) was characterized for DNA complexation (gel retardation assay, particle size, DNA release and DNase I protection), cell viability and in vitro transfection efficiency. The experiments showed that PP can effectively condense pDNA into particles. Size measurement of the complexes particles indicated that PP/DNA tended to form smaller nanoparticles than those of PEI/DNA, which was caused by the hydrophobic PBLG segments compressing the PP/DNA complex particles in aqueous solution. The representative average size of PP/DNA complex prepared using plasmid DNA (pEGFP-N1, pDNA) was about 96 nm. The condensed pDNA in the PP/pDNA complexes was significantly protected from enzymatic degradation by DNase I. Cytotoxicity studies by MTT colorimetric assays suggested that the PP had much lower toxicity than PEI. The in vitro transfection efficiency of PP/pDNA complexes improved a lot in HeLa cells, Vero cells and 293T cells as compared to that of PEI-25K by the expression of Green Fluorescent Protein (GFP) as determined by flow cytometry. Thus, the water-soluble PP copolymer showed considerable potential as carriers for gene delivery.     

Gene-activated and cell-migration guiding PEG matrices based on three dimensional patterning of RGD peptides and DNA complexes

Essential to the design of genetic bioreactors used in the human body is a consideration of how the properties of biomaterials can combine to envelope, spatially guide, reprogramme by gene transfer, and then release cells. In order to approach this goal, poly(ethylene glycol) (PEG) matrices with modulated structural features and defined spatial patterns of bioactive signals have been designed and produced. In particular, within such PEG matrices, both an adhesive RGD peptide gradient, to directionally attract NIH3T3 cells, and a designed spatial distribution of immobilized poly(ethylenimine) (PEI)/DNA complexes, to obtain a localized transfection, have been realized. These bioactive biomaterials have been designed bearing in mind that cells following an RGD gradient migrate through the matrix, in which they find the bound DNA and become transfected. Both cell migration and transfection have been monitored by fluorescence microscopy. Results show that this system is able to envelope cells, spatially guide them towards the immobilized gene complexes and locally transfect them. Therefore, the system, acting as a genetic bioreactor potentially useful for the regulation of biology at a distance, could be used to directly control cell trafficking and activation in the human body, and has many potential biomedical applications.     

Polymer-free immobilization of a cyclic RGD peptide on a nitinol stent promotes integrin-dependent endothelial coverage of strut surfaces

This study examined the utility of a stabilized cyclic RGD peptide chemically modified to selectively bind to titanium-oxide for enhanced biocompatibility of self-expanding nitinol stents. Endothelial cells express integrin receptors that promote attachment to subendothelial matrix proteins. Integrin binding to arginine-glycine-aspartic acid (RGD) peptide derivatives mimic naturally occurring adherent interactions. Irreversible covalent surface coating of conventional nitinol stents with a cyclic RGD (cRGD) peptide highly specific for integrin alpha v beta 3 might foster endothelialization after stent implantation. A selective cRGD peptide was irreversibly immobilized onto titanium oxide-rich nitinol coupons or self-expanding stents. Functionality of the engrafted RGD peptide was demonstrated using in vitro endothelial bioassays. A subsequent 7-day in vivo endothelialization study was performed using cRGD-coated self-expanding nitinol stents in rabbits. cRGD peptide coating effectively promoted endothelial cell anchorage, migration, and proliferation confirmed by increased focal adhesions. Proof-of-concept studies of rabbit cRGD stent implants showed a significant increase in endothelial coverage above stent struts relative to stents coated with BSA (cRGD = 70.1 ± 21.9 vs. BSA = 49.9 ± 21.8%, p < 0.03). Immobilization of cRGD peptides on strut surfaces represents an innovative strategy to improve endothelialization, which may facilitate vascular healing after stent implantation.     

Oligomerized alpha-helical KALA peptides with pendant arms bearing cell-adhesion, DNA-binding and endosome-buffering domains as efficient gene transfection vectors

In this study, a number of KALA-based α-helical peptides were designed and synthesized as non-viral gene carriers. The effects of lysine and histidine residues in the pendant arms and cell-binding RGD motif on DNA binding, particle size, zeta potential, cytotoxicity and gene expression efficiency were first explored. Increasing the lysine and histidine residues reduced particle size and increased zeta potential of DNA complexes, leading to greater gene expression efficiency. In addition, the introduction of RGD group further improved gene expression level. The peptide with optimal compositions, RGDN(3)K(6)H(3)CKHLAKALAKALAC (RC29), was then oligomerized to form di-, tri- and tetra-RC29 via disulfide linkage. Upon oligomerization, RC29 attained a 3-dimensional long α-helical structure with pendant arm(s) extending transversally outwards. Each arm contains a cell-adhesion motif (RGD), DNA-binding and endosome-buffering domains(.) The α-helicity of the oligomerized peptides was evaluated by circular dichroism (CD) spectroscopy, which showed that an increased oligomerization degree led to a stronger α-helical structure. These peptides form complexes with DNA efficiently. The minimum size and maximum zeta potential of tri-RC29/DNA complexes was about 200 nm and 32.5 mV, respectively. In comparison, RC29 formed DNA complexes with a similar zeta potential, but particle size was significantly larger (355 nm). DNA complexes formed at pH 7.0 yielded higher gene expressions than those formed at pH 5.5 and 6.5. Among all the oligomerized peptides, tri-RC29 provided the highest gene expression efficiency, and its peak luciferase level was 1.5 times higher than that yielded by PEI at its optimal N/P ratio (i.e. 10). Moreover, oligomerized RC29/DNA complexes were less cytotoxic than PEI/DNA complexes. These α-helical peptides can be promising carrier for delivery of therapeutic genes in the treatment of genetic disorders.     

Participation of CD11a–c/CD18 and RGD-Recognizing Adhesion Molecules in the Binding of LGL to Fibroblasts

We have previously shown that large granular lymphocytes (LGL) are inactivated by contact with natural killer (NK) resistant monolayer target cells. In this work we have analysed which adhesion molecules are involved in the binding of LGL to such targets, as exemplified by fibroblasts, and in the subsequent inhibition of their NK activity. The results indicate that antibodies against CD54 (intercellular adhesion molecule 1, ICAM-1), CD11a (leucocyte function antigen 1, LFA-1, alpha chain), and CD18 (common beta chain of the beta 2-integrin family) significantly (by 50%) reduce the binding of LGL onto inhibitory target cells. The matrix protein-based synthetic peptide RGD and anti-CD29 (the common beta chain of the beta 2-integrin family) antibodies also diminish the binding (by 35%). The effects of the antiadhesion molecule antibodies and the peptide are additive, the combination of both leading to an almost complete block of adhesion. It may be hypothesized that some of the binding-relevant adhesion molecules of the RGD-binding domain on LGL (CD29) may be involved in the delivery of the inactivating signal to the effector cell. Indeed, incubation of LGL with anti-CD11a antibodies, but neither with antibodies against other binding-relevant epitopes nor with RGD, significantly reduced their NK activity. The mechanism of the inactivation was similar to that induced by intact NK-resistant target cells. On the basis of the present results we suggest that the CD11a molecule is involved in the down-regulation of the NK activity of peripheral blood lymphocytes.     

Insulin-like growth factor binding protein-1 induces decidualization of human endometrial stromal cells via alpha5beta1 integrin

Progesterone is known to induce decidualization of human endometrial stromal cells in vitro. Decidualized stromal cells produce insulin-like growth factor binding protein-1 (IGFBP-1) as well as prolactin (PRL). In this study, we tested the possibility that IGFBP-1 directly stimulates endometrial stromal cell decidualization. Endometrial stromal cells were obtained from normal menstruating patients with uterine myoma at hysterectomy. Stromal cells were cultured for up to 4 weeks with estradiol (E(2)) and/or medroxy progesterone acetate (MPA) in the presence or the absence of IGFBP-1 and, LR(3)-IGF-I (an IGF-I analogue) that binds to the IGF-I receptor but has reduced affinity for IGFBPs. Decidualization of endometrial stromal cells was evaluated by morphological changes and PRL release into culture media. The binding of IGFBP-1 to endometrial cells was analysed using a biosensor. MPA and E(2) induced decidualization of stromal cells, while LR(3)-IGF-I inhibited decidualization by MPA and E(2) as well as PRL and IGFBP-1 secretion into medium. IGFBP-1 induced decidualization of stromal cells in the absence of MPA and E(2) in the medium. IGFBP-1-induced decidualization was not inhibited by the addition of LR(3)IGF-1 but was inhibited by the addition of an RGD peptide, however, the RGD peptide had no effect on decidualization when added alone. The binding analysis showed that IGFBP-1 bound to the surface of endometrial stromal cells and an anti-alpha5beta1 integrin antibody inhibited its binding. These results suggest that IGFBP-1 produced by endometrium can mediate progesterone-induced decidualization possibly by interacting with alpha5beta1 integrin on the surface of endometrial stromal cells.     

PEGylated guanidinylated polyallylamine as gene-delivery carrier

A novel cationic co-polymer was developed by grafting poly(ethylene glycol) (PEG) on guanidinylated polyallylamine (PAA) for gene delivery. Characterization of PEG-g-guanidinylated PAA/DNA complexes demonstrated that particle size increased and surface charge decreased with increasing the amount of PEG. The results of cytotoxicity assay proved that grafted PEG could effectively decrease the cytotoxicity of the complexes. In transfection efficiency assay, HeLa cells treated with PEG(2)-g-guanidinylated PAA (formed with 17.5 μmol guanidinylated PAA and 2 μmol PEG)/DNA (0.2 μg EGFP plasmid) complexes showed a very high level of EGFP expression. In conclusion, combination of guanidinylation and PEGylation could effectively decrease the cytotoxicity and significantly increase the transfection efficiency of PAA.     

非病毒载体聚乙烯亚胺转基因因素的优化

聚乙烯亚胺属于阳离子多聚物,可浓缩DNA作为转基因非病毒载体。但其转染影响变量较多,如果不能充分控制将不能得到重复的、良好的结果。这里测定了聚乙烯亚胺转基因效率的影响因素,为合成更复杂的人工转基因载体创造条件。我们通过聚乙烯亚胺转染编码β-半乳糖苷酶的pSVβ质粒到CO S-7和N IH 3T 3细胞中,测定质粒因素、血清、细胞密度、操作方式以及转染复合物的保存因素对聚乙烯亚胺转基因效率的影响。结果表明:质粒中生物活性抑制剂明显降低转染效率,通过截流分子量3000或10000的超滤可以除去生活性抑制剂;断裂的质粒降低转染效率;培养液中的血清、白蛋白降低转染效率;细胞密度影响转染效率;PE I/DNA复合物与细胞作用8h后吸去,转染效果最优。冻存显著降低PE I/DNA转染复合物转染效率。聚乙烯亚胺可以作为合成新型非病毒载体的骨架,但必须控制有关因素才能发挥最佳的和可重复性的转染结果。     

Gene transfer into hepatoma cells mediated by galactose-modified alpha-helical peptides

To develop a receptor-mediated gene delivery system into hepatoma cells using the cationic alpha-helical peptide as the gene carrier molecule, we modified an alpha-helical peptide, which is known to have transfection abilities into cells, with a multi-antennary ligand containing several galactose residues that provide efficient binding to the asialoglycoprotein receptor. The galactose-modified peptides formed complexes with a plasmid DNA and showed gene transfer abilities into HuH-7 cells, a human hepatoma cell line. The transfection efficiency of the peptide was increased by increasing the number of modified galactose residues on the peptide. Furthermore, considerable inhibition of the transfection efficiency by the addition of asialofetuin, which is a ligand for the asialoglycoprotein receptor, was observed in all galactose-modified peptides. Based on this result, we could confirm that the internalization of the galactose-modified peptides occurred by the receptor-mediated endocytosis pathway. In addition, to understand the transport route of the peptide-DNA complex in the cell, the effects on the transfection efficiencies with several endocytosis inhibitors were examined. As a result, it was suggested that the translocation of the peptide-DNA complex from the endocytic compartments to the cytosol mainly occurred during an early endosome step.     

Functionally active CD8alphabeta+ TCRgammadelta intestinal intraepithelial lymphocytes in athymic nu/nu mice

Murine intestinal intraepithelial lymphocytes (IEL) encompass a high proportion of TCRgammadelta cells. A vast majority of these TCRgammadelta IEL express CD8alpha, but not CD8beta (CD8alphaalpha homodimer), and are considered to develop in intestinal epithelial layers independently of a functional thymus. Here we show that TCRgammadelta cells expressing both CD8alpha and CD8beta (CD8alphabeta heterodimer) appear in athymic nu/nu mice, although their appearance is random. The IEL comprising CD8alphabeta(+) TCRgammadelta cells expressed pronounced cytolytic and IFN-gamma-producing activities after TCRgammadelta ligation, which were markedly stronger than activities of IEL lacking CD8alphabeta(+) TCRgammadelta cells. Purified CD8alphabeta(+) TCRgammadelta cells expressed strong cytolytic activities and produced large quantities of IFN-gamma after TCR engagement. CD8alphabeta(+) TCRgammadelta cells were also identified among IEL from euthymic C57BL/6 mice, although their abundance varied among individual animals. However, cytolytic and IFN-gamma-producing activities in euthymic C57BL/6 mice were markedly lower than those in athymic nu/nu mice. Our findings suggest that CD8alphabeta(+) TCRgammadelta cells can develop in the intestine independently of a functional thymus/thymic epithelial cells and that they perform biological functions in situ.     

Influence of hydroxyl groups on the biological properties of cationic polymethacrylates as gene vectors

In this study poly(aminoethyl methacrylate) (PAEMA), poly(3-amino-2-hydroxypropyl methacrylate) (PAHPMA), poly(2-(2-aminoethylamino)ethyl methacrylate) (PAEAEMA) and poly(3-(2-aminoethylamino) 2-hydroxypropyl methacrylate) (PAEAHPMA) were synthesized using atom transfer radical polymerization to evaluate the effect of hydroxyl groups on the relative properties of cationic polymeric gene vectors. The results of heparin displacement assays showed that PAHPMA possessed a stronger binding capacity than PAEMA. PAHPMA/DNA complexes and PAEAHPMA/DNA complexes had lower zeta potentials than those of PAEMA and PAEAEMA. MTT assay results indicated that PAHPMA and PAEAHPMA exhibited obviously lower cytotoxicities than PAEMA and PAEAEMA. Subsequently, in vitro gene transfection studies in 293T cells without serum showed that PAHPMA exhibited a lower transfection efficiency than PAEMA and PAEAHPMA/DNA complexes possessed a similar transfection efficiency to PAEAEMA/DNA complexes. Moreover, PAHPMA and PAEAHPMA retained similar transfection efficiencies in DMEM with 10% serum, but PAEMA and PAEAEMA showed slightly lower transfection efficiencies than in the absence of serum. The reason for these phenomena might be attributed to the introduction of hydroxyl groups into PAHPMA and PAEAHPMA, i.e. the existence of hydroxyl groups might increase the binding capacity to DNA and at the same time decrease the surface charge of the polymer/DNA complexes due to the formation of hydrogen bonds between the polymers and DNA. Therefore, a lower zeta potential and stronger binding ability may result in a lower gene transfection efficiency. This effect of hydroxyl groups decreased with increasing amino group density on the polymer.     

Cellular recognition of synthetic peptide amphiphiles in self-assembled monolayer films

The incorporation of lipidated cell adhesion peptides into self-assembled structures such as films provides the opportunity to develop unique biomimetic materials with well-organized interfaces. Synthetic dialkyl tails have been linked to the amino-terminus, carboxyl-terminus, and both termini of the cell recognition sequence Arg-Gly-Asp (RGD) to produce amino-coupled, carboxyl-coupled, and looped RGD peptide amphiphiles. All three amphiphilic RGD versions self-assembled into fairly stable mixed monolayers that deposited well as Langmuir-Blodgett films on surfaces, except for films containing amino-coupled RGD amphiphiles at high peptide concentrations. FT-IR studies showed that amino-coupled RGD head groups formed the strongest lateral hydrogen bonds. Melanoma cells spread on looped RGD amphiphiles in a concentration-dependent manner, spread indiscriminately on carboxyl-coupled RGD amphiphiles, and did not spread on amino-coupled RGD amphiphiles. Looped RGD amphiphiles promoted the adhesion, spreading, and cytoskeletal reorganization of melanoma and endothelial cells while control looped Arg-Gly-Glu (RGE) amphiphiles inhibited them. Antibody inhibition of the integrin receptor alpha3beta1 blocked melanoma cell adhesion to looped RGD amphiphiles. These results confirm that novel biomolecular materials containing synthetic peptide amphiphiles have the potential to control cellular behavior in a specific manner.